Nearly all licensed vaccines protect through antibodies rather than cell-mediated immunity. A critical aspect of vaccine-induced serological protection is the duration of antibody titer post-boost. Our basic understanding of T-cell dependent antibody responses involves presentation of antigens by dendritic cells to specific CD4+ T cells, which then proliferate and differentiate into helper T cells (Tfh) that engage antigen-engaged B cells that have moved to the T-B border of secondary lymphoid tissues (spleen or lymph node). These interactions can generate a population of short-lived, high-rate antibody secreting cells (the extrafollicular antibody response), which contributes to acute host defense but not to long-lived protection. The latter involves migration of antigen-activated T cells and the associated antigen-specific B cells into the B cell follicle where they set up the germinal center (GC) reaction. Here continued T-B interaction leads to somatic hypermutation and isotype class switching, producing antibodies of higher affinity and different effector class, while generating both memory B cells and also plasmablasts that can become long-lived plasma cells (LLPC) if they reach the proper niche, which is mainly but not exclusively located in the bone marrow. While this general outline is well-established, the molecular signals that guide the engagement of T and B cells to generate a maximally productive response, the role of Tfh cells in determining the choice between memory B cells and LLPCs, and what determines how plasmablasts become LLPCs remain unclear. This project is a collaborative effort among laboratories with expertise in vaccine development, adjuvant function, and cellular immunology that aims to examine how variations in the quality and quantity of Tfh, stimuli for B cells, and GC output contribute to the magnitude and durability of antibody responses post-vaccination. In FY17, we made significant progress towards evaluating the contribution of carrier determinants and adjuvants to promoting high-titer, long-lived antibody responses against Pfs25, a leading TBV candidate for Plasmodium falciparum. We determined that both adjuvants and carrier proteins influence the magnitude and capacity of Pfs25-specific humoral responses to remain above a protective level. Additionally, we found that a liposomal adjuvant formulation with a TLR4 agonist and QS21, GLA-LSQ, profoundly impacted the magnitude of the Tfh and LLPC response against Pfs25, an effect that was further enhanced following conjugation to antigenic carrier proteins. Importantly, this adjuvant-dependent Tfh cell priming correlated with a large LLPC response and durable, functional antibody response. A manuscript summarizing these findings was published in Scientific Reports. We established a Research Collaboration Agreement with the Walter Reed Army Institute of Research (WRAIR) to obtain potent adjuvants with clinical potential. We are using these Army Liposome Formulation (ALF)-based adjuvants, in combination with carrier-specific tetramers to relate Tfh quality and quantity to vaccine outcome. We are also particularly interested in whether different adjuvants promote CD4+ T cells to adopt a suppressive T follicular regulatory (Tfr) phenotype and, if so, where and when do these differentiation events occur in lymphoid organs. To address these questions, we are employing multi-parameter flow cytometry and advanced imaging techniques to quantify the kinetics and location of Tfh and Tfr differentiation following vaccination. We found that the ALFQ adjuvant induced a higher Tfh:Tfr cell ratio as compared to two alum containing adjuvants, alhydrogel and ALFQA. These qualitative differences in T helper differentiation correlated with larger GC and plasmablast responses after secondary immunization. Interestingly, Tfr cells were nearly absent from active GC reactions and were, instead, found at the T-B border in all the immunization groups examined, raising several questions about how Tfr cells regulate Tfh and GC B cells. Related findings about Tfr localization are emerging from separate collaborative studies using human lymph nodes. We recently demonstrated that immunization with peptide or protein antigens emulsified in oil and surfactant-only containing water-in-oil adjuvants such as IFA (Incomplete Freunds Adjuvant) results in polarized Tfh responses (Riteau et al. JI, 2016). Using the same immunization protocol, we carefully characterized antigen specific T follicular regulatory (Tfr) cells by flow cytometry and a t-distributed stochastic neighbor embedding (t-SNE) algorithm, which represent between one and five percent of the whole antigen specific population. Since we published that type I IFN receptor (Ifnar) deficient mice display reduced antigen specific Tfh responses, we hypothesized that this could lead to a reduced humoral response. Surprisingly, despite displaying decreased frequencies of GC B cells, IFA-immunized Ifnar-/- showed no impairment in serum IgG antibody titers as compared to wild-type immunized animals. Further investigation revealed that while mounting defective Tfh responses, IFA-immunized Ifnar-/- mice exhibit a significant increase in the frequency of antigen specific Tfr cells. Unexpectedly, using a cytokine reporter system, we demonstrated the production of IL-21, a cytokine critical for the generation of GC B lymphocytes, by this Tfr population ex vivo. Moreover, IFA-immunized IL-21 receptor deficient animals while showing impaired GC formation, displayed decreased frequencies of Tfr cells but surprisingly, an unaltered Tfh response. These findings indicate that in water-in-oil adjuvant immunization, type I IFN deficiency results in the increased generation of Tfr cells which we propose can promote B cell maturation and antibody production through IL-21 signaling. We are currently employing multiparameter confocal imaging to visualize IL-21 producing cells in the draining lymph nodes of IFA-immunized IL-21 reporter mice. A key aim of this project is the identification of vaccine formulations that will increase the duration of the antibody response against malaria vaccine candidates. Our prototype target malaria antigen is Pfs25, which is undergoing Phase 1 trials in humans as a Pichia-expressed recombinant protein conjugated to the carrier protein ExoProtein A (EPA) expressed in E. coli with a molar ratio of 3:1, and formulated with the commercially available adjuvant Alhydrogel: Pfs25-EPA/Alhydrogel. Pfs25-EPA/Alhydrogel is undergoing Phase 1 trials in malaria-nave volunteers in the US (dose-escalating trial) and malaria-experienced volunteers in Mali (dose-escalating; double-blinded; placebo-controlled trial). Sera collected from volunteers in the US and Mali are being assessed for seroreactivity by standardized ELISA, and transmission-blocking antibody activity is measured in membrane feeding assays. Serum antibody levels in either assay are being measured before and after each vaccine dose, and then periodically thereafter to assess the duration of the antibody response, including the functional antibody response. In Mali, mosquitoes are fed directly on vaccinees to determine their infectivity/malaria transmission potential, and this will be related to the antibody measurements. The Pfs25-EPA/Alhydrogel product will be a benchmark against which we will compare novel Pfs25 products and formulations in our animal studies. Our animal studies of adjuvants to date support our plan to test Pfs25-based conjugate vaccines using alternative adjuvants in humans, and our initial focus is on the commercial product AS01 from GSK and similar liposomal formulations from IDRI and WRAIR that have not been in the clinic.